Functional characterization of an IgE-class monoclonal antibody specific for the bullous pemphigoid autoantigen, BP180.

BP180 (collagen XVII) is the target antigen in several autoimmune diseases including bullous pemphigoid (BP). Both IgE and IgG class autoantibodies have been shown to be pathogenic in BP; however, studies designed to elucidate the patho-mechanisms mediated specifically by the IgE-class autoantibodies are limited by the low levels (ng/mL) of IgE in human sera. In this report, we developed mouse IgE class monoclonal antibodies (MAbs) against the immunodominant NC16A domain of the human BP180 protein and characterized two of the resultant MAbs, designated 395A5 and 395D2. Epitope mapping studies revealed that both MAbs target segment 2 of NC16A, as was described for IgE and IgG class BP autoantibodies. Also similar to BP IgE, MAb 395A5 showed indirect immunofluorescence labeling of the basement membrane zone (BMZ) of human skin, stimulated histamine release from mast cells when triggered with NC16A, and induced keratinocyte production of IL-8. The 395D2 MAb was also able to trigger antigen-specific histamine release from mast cells; however, in contrast to BP IgE and 395A5, 395D2 did not label the cutaneous BMZ, nor did it induce IL-8 production in keratinocytes. In summary, these studies underscore the importance of functionally characterizing MAbs generated for use in human disease models. The 395A5 IgE class murine MAb was shown to share several key functional properties with the pathogenically active IgE produced by BP patients. We therefore expect that this MAb will prove to be a useful tool for dissecting the mechanisms used by BP180-NC16A-specific IgE antibodies in the induction of BP skin lesions.

Exploring the molecular origins of protein dynamics in the active site of human carbonic anhydrase II.

We present three-pulse vibrational echo measurements of azide ion bound to the active site Zn of human carbonic anhydrase II (HCA II) and of two separate active-site mutants Thr199 --> Ala (T199A) and Leu198 --> Phe (L198F). Because structural motions of the protein active site influence the frequency of bound ligands, the differences in the time scales of the frequency-frequency correlation functions (FFCFs) obtained from global fits to each set of data allow us to make inferences about the time scales of the active site dynamics of HCA II. Surprisingly, the deletion of a potential electrostatic interaction in results in very little change in the FFCF, but the insertion of the bulky phenylalanine ring in causes much faster dynamics. We conclude that the fast, sub-picosecond time scale in the correlation function is attributable to hydrogen bond dynamics, and the slow, apparently static contribution is due to the conformational flexibility of Zn-bound azide in the active site.